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J Appl Physiol 97: 2176-2183, 2004. First published August 6, 2004; doi:10.1152/japplphysiol.00985.2003
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Insulin-like growth factor-2 genotype, fat-free mass, and muscle performance across the adult life span

Matthew A. Schrager,1,2 Stephen M. Roth,1 Robert E. Ferrell,3 E. Jeffrey Metter,2 Estelle Russek-Cohen,4 Nicole A. Lynch,5 Rosemary S. Lindle,1 and Ben F. Hurley1

1Department of Kinesiology, College of Health and Human Performance, 4Department of Animal & Avian Sciences, Biometrics Program, University of Maryland, College Park 20742; 2National Institute on Aging, Gerontology Research Center, Baltimore, Maryland 21224; 3Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; and 5Division of Gerontology, University of Maryland School of Medicine, Baltimore Veteran's Affairs Medical Center, Baltimore, Maryland 21201

Submitted 11 September 2003 ; accepted in final form 3 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The influence of insulin-like growth factor-2 (IGF2) genotype on total body fat-free mass (FFM), muscle strength, and sustained power (SP) was evaluated repeatedly at ~2-yr intervals in two cohorts from the Baltimore Longitudinal Study of Aging. Cohort 1 was comprised of 94 men tested for isometric grip strength and SP. Cohort 2 was comprised of 246 men and 239 women tested for total body FFM and isokinetic peak torque. Subjects were retrospectively genotyped for the IGF2 gene's ApaI polymorphism. Differences between genotype groups for total FFM, strength, and SP at first visit, at peak age (35 yr), at age 65, and across the adult age span were analyzed using either two-sample t-tests or mixed-effects models, depending on the specific comparisons made. Isokinetic arm strength at the time of first visit was lower in A/A men than in G/G men (P < 0.05). Compared with G/G women, A/A women had lower total body FFM, lower isokinetic arm and leg strength at the time of first visit, and lower values at age 35 (all P < 0.05) for these muscle phenotypes. Furthermore, this difference between the genotype groups was maintained at age 65 and across the adult age span (P < 0.05). No genotype-associated differences in rates of loss of grip strength or SP were found in cohort 1. These results from cohort 2 support the hypothesis that variation within a gene known to influence developing muscle affects muscle mass and muscle function in later life.

muscle mass; muscle strength; muscle power; genetics; gender

AGE-ASSOCIATED LOSSES IN MUSCLE MASS, strength, and power (sarcopenia) have detrimental effects on functional capacity, rates of injury, morbidity, and mortality (4, 9, 36, 37). Heritability studies have found genetic contributions of up to 66 and 65% for muscle mass (1) and strength (47), respectively. In addition, recent studies have found significant relationships between prenatal growth (birth weight) and adult muscle mass (13, 23, 42) and grip strength (27, 50) and have shown midlife strength to be a predictor of mortality (45). Despite these relationships, little research has addressed the influence of specific candidate genes on muscle mass or muscle strength and body composition. However, significant influences of polymorphisms of one such candidate gene, insulin-like growth factor (IGF)-2 (IGF2), on body composition (41), birth weight, and grip strength in middle age have been reported (51) (muscle mass or power differences were not examined in these investigations). Furthermore, because fiber number is established at or immediately after birth in mammals (16), it has been suggested that those who have skeletal muscle hypoplasia at birth may be predisposed to potentially debilitating losses in muscle mass and strength with advancing age (27). Therefore, there is a need to examine the potential influence of IGF2 polymorphism on muscle mass and muscle performance at ages before and after the beginning of age-associated losses in these muscle phenotypes (e.g., 35 and 65 yr, respectively) (30) and across the entire adult life span.

Given the role of IGF-II in satellite cell proliferation (19) and the age-associated decrease in IGF2 gene expression in response to muscle damage (35), it is possible that IGF-II could influence acute regenerative capacity in aging human muscle in a way that the cumulative effect of these diminished responses to exercise or to increasing oxidative stress with age (18) could influence rates of age-associated losses in muscle mass and strength in humans (22).

The two IGF2 genotype studies mentioned above have demonstrated lower body mass and body mass index (BMI) (41) and grip strength (51) in men homozygous for the rare A allele of the 3'-untranslated region ApaI polymorphism. Based on these findings (41, 51) and the suggested influence of IGF-II on adult body weight (31), and given the established relationships between body weight and fat-free mass (FFM) across the adult age span (11), we hypothesized that men and women homozygous for the A allele at the IGF2 ApaI locus would have significantly lower muscle mass, strength, and power at peak age (35 yr) (30) than men and women homozygous for the common G allele. Moreover, this difference between genotype groups would be maintained at age 65 and across the adult age span. Given the evidence of the role of IGF-II in muscle regeneration and previous findings showing greater body mass (41) and grip strength (51) in middle-aged IGF2 ApaI G homozygous men, our secondary hypothesis was that men and women homozygous for the A allele would have significantly greater rates of loss of muscle strength and power with age.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects.   Subjects were volunteers in the Baltimore Longitudinal Study of Aging (BLSA), an ongoing multidisciplinary observational study initiated in 1958. The BLSA has employed various measures of muscle mass and function during specific periods in the study's history. The two cohorts for the present study are defined on the basis of the differing muscle mass and function assessments made during two periods: 1) 1960–1985 (cohort 1) and 2) 1992–2002 (cohort 2). Cohort 1 (n = 94, age range = 22–80 yr) was comprised only of Caucasian men. Cohort 2 included both men and women (n = 485; 246 men, 239 women, age range = 20–94 yr) and consisted of 403 Caucasians (196 men, 207 women), 70 African-Americans (46 men, 24 women), and 12 people (4 men, 8 women) of other races. In addition, cohort 1 was significantly younger than cohort 2 at time of first visit (Tables 1 and 2). BLSA participants are community-residing, healthy volunteers who tend to be well educated, with above-average income and access to medical care. Subjects visited the Gerontology Research Center at 1- to 2-yr intervals for 2 days of medical, psychological, and physiological testing, which includes assessments of FFM, muscle strength, and sustained power (SP). The average number of years between first and last tests in the BLSA, and consequently the average total number of biennial tests, was significantly greater per subject in cohort 1 compared with cohort 2. Specifically, an average period of 12.7 ± 6.2 yr (range of 2–25 yr) in cohort 1 and 4.5 ± 2.0 yr (range of 0–10 yr) in cohort 2 elapsed between first and last visits, which corresponded to an average of 5.3 ± 2.5 visits (range of 2–10 visits) in cohort 1 and 1.9 ± 0.8 visits (range 1–5 visits) in cohort 2. Many subjects were not evaluated over the entire period (either 1960–1985 or 1992–2002), which resulted in fewer visits per subject than would otherwise be predicted based on 1- to 2-yr intervals throughout the entire period. Although the repeated measures in cohort 2 likely improve the reliability of the muscle mass and performance measures and increase statistical power, the relatively short duration of follow-up in this cohort is not sufficient for the thorough analysis of potential longitudinal or aging effects on muscle phenotypes. The cross-sectional analysis of data from cohort 2 assesses associations between IGF2 ApaI genotype and relationships between age and FFM, as well as muscle strength and SP. In contrast, the longitudinal data from cohort 1 contain sufficient follow-up for the detection of aging effects on muscle phenotypes of interest, so that the associations with IGF2 ApaI genotype can be determined. In addition, the repeated follow-up assessments in cohort 1 serve to improve the stability of the measurements to determine aging-related rates, as well as comparisons at specific ages. In this regard, many previous longitudinal studies contain observations at only two time points (12, 46). Thus, by analyzing results from both cohort 1 and cohort 2 collectively, we believe that we can better test our primary hypothesis. To have a sufficient follow-up to be able to detect changes in strength or SP with aging, only those subjects in cohort 1 who had at least 7 yr of follow-up [n = 80; mean follow-up = 14.2 ± 5.4 yr (range of 7–26 yr); mean number of visits = 5.8 ± 2.4 visits (range of 2–10 visits)] were analyzed to test our secondary hypothesis (i.e., IGF2 ApaI genotype affects aging-associated rates of loss in muscle strength and SP). Physical characteristics of the subjects with at least 7 yr of follow-up did not differ significantly from the entire cohort.


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  		Table 1. Cohort 1 physical characteristics by IGF2 genotype at time of first visit

 

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  		Table 2. Cohort 2 physical characteristics, including FFM, by IGF2 genotype and gender at time of first visit

 
All subjects in the present study received a complete medical history and physical examination at each visit, and those with clinical cardiovascular or musculoskeletal disease that potentially could be aggravated by the strength and SP testing procedures were excluded. Detailed exclusionary criteria have been presented previously (30, 33). The experimental protocols were approved by the Institutional Review Boards for Human Subjects at Johns Hopkins Bayview Medical Center (Baltimore, MD), the University of Maryland (College Park, MD), and the University of Pittsburgh (Pittsburgh, PA).

Physical activity.   Leisure-time physical activity (LTPA) was self reported and based on the amount of time spent performing 97 activities since the previous BLSA visit. The reported time spent performing daily activities was based on a routine day, and the intensities of reported activities were expressed in metabolic units (METs) based on the coding scheme described by Ainsworth et al. (2). The number of minutes spent performing each activity was multiplied by the appropriate MET value to obtain a value for total MET minutes/day. Further details of the administration of this questionnaire in the BLSA have also been outlined previously (36, 37).

Total body mass and FFM.   Body weight was measured to the nearest 0.1 kg by using a medical beam scale (Detecto, Webb City, MO), and height was measured to the nearest 0.5 cm. A subset of cohort 2 (n = 354; 148 men, 206 women) was assessed for total body FFM using a dual-energy X-ray absorptiometry (DEXA) scanner (model DRX-L, LUNAR Radiation, Madison, WI). All DEXA scans were analyzed using LUNAR software version 3.6/1.3y program for body composition analysis (LUNAR) (32). Based on the total body DEXA scan, nonosseous total body FFM (estimated muscle mass) was determined (30). Reliability was assessed by performing two total-body scans, 6 wk apart on 12 older (>65 yr) subjects, and DEXA calibration was done daily as described (33).

Measurement of strength and SP.   Isometric grip strength and SP data collected during the period of 1960–1985 (cohort 1) and the associated measurement procedures have been described in other BLSA studies (24, 36). Women did not enter the BLSA until 1978, and because only one woman in this sample also had IGF2 ApaI genotype data available, the analyses for cohort 1 were limited to men. Briefly, isometric grip strength was measured using the Smedley hand dynamometer (Stoelting, Wood Dale, IL). Subjects were in a seated position with the tested arm extended on a table during testing. Three trials separated by brief rest periods were performed using each hand alternately. Subjects were encouraged to exert their maximal grip, and the best result for each hand was chosen. For the present study, grip strength of only the dominant hand was analyzed for comparison to a recent short report on the influence of IGF2 ApaI genotype on grip strength (51). Arm SP was also measured in cohort 1, as described previously (52), using a bicycle converted to act as a drive shaft to power a calibrated automobile generator. Subjects were recumbent on a reinforced bed that limited excess bed movement, and the apparatus allowed for a full range of motion at the elbows. They were instructed to perform a maximal effort for 10–15 s of arm cranking at each of four load settings (1–4 A), and the maximal scale reading was converted to units of power (kg/min) by using a calibration curve. For the present study, only the values obtained at the 1- and 4-A settings were presented. For repeated SP tests and grip strength tests, there was a 6% coefficient of variation (24).

In cohort 2 (1992–2002), an isokinetic dynamometer [Kinetic Communicator (Kin-Com) model 125E Plus, Chattecx, Chattanooga, TN] was used to measure concentric and eccentric peak torque (PT) in the BLSA. The words "shortening" and "lengthening" are substituted throughout the present study for the more commonly used terms "concentric" and "eccentric," respectively, based on the recommendations of Faulkner (8). Shortening and lengthening PT (PTS and PTL, respectively) were measured in the dominant elbow flexors and elbow extensors at an angular velocity of 0.79 rad/s (45°/s), and PTS and PTL were measured in the dominant knee flexors and knee extensors at angular velocities of 30°/s (0.52 rad/s) and 180°/s (3.14 rad/s). To have a more manageable number of strength measures, the following summed values were created: arm PTS (elbow flexor PTS + elbow extensor PTS), arm PTL (elbow flexor PTL + elbow extensor PTL), and leg PTS (knee flexor PTS + knee extensor PTS at angular velocity of either 30°/s or 180°/s). For each test, subjects performed three submaximal practice repetitions followed by three maximal efforts, with the maximal efforts separated by 30-s rest intervals. Of the three maximal trials, the one yielding the highest PT was identified and used in the analyses. A more detailed description of subject positioning and stabilization, warm-up, and testing order, along with gravity compensation and calibration of the Kin-Com has been outlined previously (30, 33).

Genotype.   Genomic DNA was extracted from whole blood samples using standard procedures (38), and genotyping of the IGF2 ApaI polymorphism was done using PCR and ApaI restriction digest of the PCR product, as previously described (41). Subjects were categorized as exhibiting the A/A, G/A, or G/G genotype, and direct sequencing was used to confirm the accuracy of the genotyping methods.

Statistical methods.   Statistical analyses were completed using SAS software version 8.2 (SAS Institute, Cary, NC). Each gender was analyzed separately. Differences between genotype groups in physical characteristics, and total FFM, strength, and SP at first visit (i.e., baseline) were analyzed using two-sample t-tests (Tables 1 and 2). Linear mixed-effects regression models for repeated measures have been described previously (56) and were used to analyze the potential associations between IGF2 ApaI polymorphism and FFM, strength, or SP at peak age by comparing model-predicted genotype group means at 35 yr, with advancing age by comparing model-predicted genotype group means at age 65, and across the adult age span by comparing overall genotype group means, after adjustment for known effects of age, body size, race, physical activity, and strength training (30, 33, 36, 48, 54). The models used permit the inclusion of both time-dependent (e.g., body weight) and time-independent (e.g., genotype) covariates. We considered two-way interactions and, in particular, were interested in a potential for an age-by-genotype interaction. Such an interaction could indicate a more rapid decline with age for one genotype compared with another genotype. The spatial power correlation structure applied in this study fit the data best by using Akaike's information criterion (56). This structure assumes that observations within a subject are correlated and that this correlation declines with increasing time between observations. Observations recorded on different subjects are regarded as independent (i.e., uncorrelated). This correlation structure accounts for the presence of unequally spaced observations in time, which are fairly common in the BLSA due to missed or delayed visits, and allows one to include all of the data collected.

Multicollinearity among explanatory variables was reduced by centering age, height, weight, and BMI for each gender (i.e., the corresponding means were subtracted from each of the individual values) (26). Because the IGF2 gene is maternally imprinted in humans (40) and, therefore, only the paternal allele is expressed, we also performed the linear mixed-effects regression analyses on only the homozygous individuals (A/A and G/G) to examine the associations of each allele with FFM, strength, and SP. Backward elimination of statistically nonsignificant terms (P > 0.05) from the full model was used. Finally, to test the goodness of fit of the model and possible effects of influential observations, the procedure outlined by Lesaffre and Verbeke (29) was used. Three influential observations in the SP measure were detected and omitted due to their effect on the results.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tables 1 and 2 illustrate the similarity in physical characteristics among the three genotype groups at the time of first BLSA visit (i.e., baseline) for cohorts 1 and 2, respectively. The only significant difference was that women in the G/A genotype group were younger than the A/A and G/G genotype groups (P < 0.05; Table 2). In addition, the LTPA questionnaire revealed that a significant percentage of subjects participated in regular strength training and that this percentage increased between the periods of 1960–1985 (cohort 1) and 1992–2002 (cohort 2). Specifically, in cohort 1, 14.9% of subjects reported the equivalent of at least two weekly sessions of ≥20 min of strength training compared with 21.2% of the men and 18.6% of the women in the comparatively older cohort 2. This finding may reflect an increased awareness within the general population of the health benefits of strength training recently described in elderly populations (9, 20) or a varying perception of physical activity in the two cohorts. Among genotype groups, there were no significant differences in the number of subjects who participated in strength training or in the time spent strength training. Of the 94 Caucasian men in cohort 1, 13 were A homozygotes, 32 were heterozygotes (G/A), and 49 were G homozygotes. The G allele frequency in this sample was 69.1%, and the A allele frequency was 30.9%. The observed genotypic frequencies of this sample were in Hardy-Weinberg equilibrium ({chi}2 analysis, P > 0.05). Of the 246 men and 239 women observed in cohort 2 (Table 2), 57 were A homozygotes, 190 were heterozygotes, and 238 were G homozygotes. The G allele frequency in this sample was 68.7%, and the A allele frequency was 31.3%. Within each gender, the genotype frequencies were in Hardy-Weinberg equilibrium for women but not for men in cohort 2 ({chi}2 analysis, P < 0.05). Finally, African-American subjects in this sample (n = 70; 46 men, 24 women) were in Hardy-Weinberg equilibrium within each gender ({chi}2 analysis, P > 0.05). In cohort 1, there were no significant differences among genotype groups for levels of strength or SP at time of first visit. However, Figs. 1 and 2 illustrate several significant differences among genotype groups at time of first visit for isokinetic arm and 30°/s leg PT, respectively. For example, A/A men had significantly lower isokinetic arm PTS than G/G men (P < 0.05), and A/A women had significantly lower isokinetic arm PTS and PTL than G/G and G/A women (P < 0.05) (Fig. 1). The A/A women also had significantly lower 30°/s isokinetic leg PTS than the G/G women (Fig. 1). Consistent with the latter strength findings, A/A women also had significantly lower total FFM than both G/A and G/G women at the time of first visit (P < 0.05; Table 2).



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  		Fig. 1. Shortening (S) and lengthening (L) peak torque in the arm at first visit in A/A (n = 32), G/A (n = 95), and G/G (n = 119) men and A/A (n = 25), G/A (n = 95), and G/G (n = 119) women in cohort 2. Values are means ± SE. *Significantly lower than G/G genotype group (P < 0.05). {dagger}Significantly lower than G/A and G/G genotype groups (P < 0.05).

 


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  		Fig. 2. Shortening peak torque in the leg at 30°/s at first visit in A/A (n = 32), G/A (n = 95), and G/G (n = 119) men and A/A (n = 25), G/A (n = 95), and G/G (n = 119) women in cohort 2. Values are means ± SE. *Significantly lower than G/G genotype group (P < 0.05).

 
As determined by backward elimination, the final mixed-effects regression model for FFM in men included the following terms: height, age, age2, genotype, an age-genotype interaction, an age-height interaction, and time spent strength training (min/day). In women, the final FFM model included height, age, age2, genotype, an age-height interaction, and time spent strength training. For strength and power measures, the final model for men included BMI, age, age2, genotype, and time spent strength training, whereas for women it included BMI, age, genotype, age-BMI, and time spent strength training. Overall LTPA level did not remain in any of the models after the backward elimination procedure. Using these final mixed-effects regression models, we found no significant genotype main effects for FFM, strength, or power in men when either all three or only the homozygous individuals (A/A and G/G) were included in the analyses. However, there were significant main effects for genotype in women for isokinetic arm PTS and arm PTL when all three genotypes were included (P < 0.05). When only the homozygous (A/A and G/G) were analyzed, there were significant main effects for genotype for total FFM, isokinetic arm PTS, arm PTL, and 30°/s leg PTS (P < 0.05). These findings were consistent with the significant baseline findings in Figs. 1 and 2 in that, for each of the measures, the model-predicted means were all greater in the G/G women than in the A/A women, thus supporting our primary hypothesis. Moreover, the hypothesis that IGF2 ApaI genotype is associated with peak (i.e., at age 35) total FFM and muscle strength and SP was supported by the results in Tables 3 and 4 in women. Specifically, when only the homozygous individuals were compared, G/G women demonstrated greater model-predicted means at age 35 for total FFM and for each one of the three strength measures: isokinetic arm PTS and PTL and leg PTS (P < 0.05). Furthermore, this difference between A/A and G/G women was maintained at age 65 (Table 4) for each of the above measures. Finally, results similar to those above were found for leg PTS at 180°/s (Tables 3 and 4) and when the summed strength measures above were divided into flexor- and extensor-specific measures (30, 33).


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  		Table 3. Adjusted mean values at age 35 for FFM, strength, and power measures by IGF2 genotype and gender in cohorts 1 and 2

 

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  		Table 4. Adjusted mean values at age 65 for FFM, strength, and power measures by IGF2 genotype and gender in cohorts 1 and 2

 
Contrary to our secondary hypothesis, rates of change in grip strength and SP with aging were not significantly associated with the IGF2 ApaI genotype. For the men in cohort 1 with at least 7 yr of follow-up, the mean rates of loss were the following. For grip strength, A/A men lost 3.7%, G/A men lost 2.6%, and G/G men lost 1.7% per decade whereas for SP A/A men lost 7.5%, G/A men lost 4.4%, and G/G men lost 3.3% per decade. However, when either all three genotype groups or only the homozygous individuals were compared, the differences in rates of loss between genotype groups were not statistically significant for either grip strength (P = 0.16) or SP (P = 0.40). In the analyses of total FFM and the three measures of isokinetic strength in cohort 2, the only significant age-genotype interaction was for total FFM in men (P < 0.05), which indicated that G/G men showed a greater age-associated loss in FFM than A/A men across the adult age span. Finally, the analyses in Caucasian subjects only in cohort 2 revealed similar results to those presented above for the entire cohort 2 for all muscle phenotypes.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study partially support the primary hypothesis that individuals homozygous for the A allele at the IGF2 ApaI locus possess lower FFM, strength, and SP at age 35, at age 65, and across the adult age span compared with individuals homozygous for the G allele. Isokinetic arm strength (arm PTS) at the time of first visit was significantly lower in A/A men compared with G/G men. Greater support for our primary hypothesis was observed in women than in men. For example, A/A women had lower total body FFM, lower isokinetic arm strength during both the shortening and lengthening phases of exertion, and lower leg strength during the shortening phase of exertion (at contraction velocities of both 30 and 180°/s) than G/G women at the time of first visit and at age 35. This difference between the genotype groups was maintained at age 65 and across the age span. However, the results did not support the secondary hypothesis that A/A men would demonstrate greater rates of age-associated decline in grip strength or SP than G/G men. The only genotype-associated significant difference in rates of loss with age was found with G/G men demonstrating an unexpected greater rate of loss in FFM compared with A/A men. None of the analyses in women included any genotype-associated significant differences in rates of loss. This significant finding in men, however, must be viewed with caution because BLSA protocols dictate that men receive relatively fewer DEXA scans; therefore, very little follow-up FFM data exists, making estimates of rates of change with aging less accurate in this sample compared with measures with greater follow-up (i.e., grip strength and SP).

The role of IGF-II in modulating muscle mass during fetal development has been clearly demonstrated in gene manipulation studies in mice (7, 28). For example, DeChiara et al. (7) showed that disrupting the function of the IGF2 gene via targeting results in a growth-deficiency phenotype. Moreover, previous studies have used controlled intercrosses and quantitative trail loci mapping analysis to demonstrate that polymorphism in the IGF2 region of chromosome 2 has direct effects on muscle deposition and adult muscle mass in pigs (21, 39). Given that IGF2 is highly conserved between this region in pigs and human chromosome 11p (21), it is possible that polymorphism in the IGF2 gene region also has an effect on adult muscle mass in humans.

The importance of genetic influences on birth weight and muscle mass has been somewhat downplayed (6, 27). In one study, weights of babies born after ovum donation were strongly associated with the weights of the recipient mother (an environmental factor) but were not associated with the weights of the female donors (a genetic factor) (5). Because IGF2 is maternally imprinted, an IGF2 allele is inherited from the father and not from the mother. Thus epidemiological studies limited to maternal genetic influences (5, 6, 27) would not sufficiently account for the effect, already demonstrated in mice (7) and pigs (21, 39), of a paternally inherited candidate gene such as IGF2 on human muscle development in utero or for the potential effect on adult muscle mass.

A hypothesis recently put forth by Kuh et al. (27) to explain their recent finding of a positive relationship between birth weight and grip strength in middle age in humans states that the number of muscle fibers present at birth may influence the aging-associated losses in muscle strength in adult life. Specifically, individuals born with fewer muscle fibers (as a result of genetic and/or environmental factors in utero) would be at a considerable disadvantage for withstanding the inevitable losses in muscle fibers that characterize the aging process. A study of piglets has shown that an environmental factor, poor fetal position for placental nutrient delivery, is associated with lower numbers of muscle fibers at birth (58). Wigmore and Stickland (57) stated that such a deficit likely persists into adulthood because fiber hyperplasia was found to cease between 85 and 95 days of gestation in a related study from the same group. Considering that IGF-II stimulates in vitro somite myogenesis to increase fiber number (44) and that the IGF2 ApaI polymorphism exerts an independent effect on middle-age grip strength (51), a genetic factor such as IGF2 polymorphism may similarly have an influence on fiber number and, therefore, muscle mass at birth that persists across the adult age span. There is some support for a potential link between the developmental role of the IGF2 gene region and its association with muscle phenotypes in adults. This comes from studies linking the intrauterine period and body size and composition (13), lean mass (13, 43), and muscle performance (27, 51) in later life in humans, together with the above hypothesis proposed by Kuh et al.

In addition, animal studies in muscle development have suggested mechanisms by which IGF-II affects myogenesis (10, 44), and these mechanisms may have effects on muscle mass and performance in later life. For instance, IGF-II acts in conjunction with IGF-I to stimulate myoblast differentiation via increased expression of the transcription factor myogenin (10). A more recently elucidated role of IGF-II in muscle development is as a survival factor for proliferating myoblasts. Stewart and Rotwein (53) demonstrated that exogenous IGF-II prevents inappropriate cell death of primary myoblasts in their transition to terminally differentiated myoblasts. Finally, in the above in vitro experiments that demonstrate the role of IGF-II in myoblast proliferation and differentiation, the effects of varying levels of IGF-II gene expression and/or protein concentration are highlighted as potential mechanisms of muscle growth.

Normal maintenance of muscle mass and function involves repeated cycles of injury and satellite cell-mediated repair (19), and age-associated decrements in this repair process have also been shown to be associated with levels of IGF gene expression. For example, Marsh et al. (35) compared young, adult, and old rats exposed to bupivicaine-induced muscle damage and showed that young rats exhibit relatively higher levels of IGF-II expression and better recovery of muscle mass and protein concentration postinjury. Barton-Davis et al. (3) additionally reported a major effect of directed overexpression of IGF-I on the aging-associated losses in strength. Although we did not show a significant effect of IGF2 ApaI polymorphism on the rates of loss of grip strength or SP in the analysis of cohort 1, the difference in rates of loss of SP between A/A and G/G men in this cohort was fairly large. Perhaps the small sample size in this cohort, especially in the A/A group (n = 13), prevented this difference from reaching significance. Therefore, due to this sample size and the significant age-genotype interaction in cohort 2 men, it appears that further exploration of our secondary hypothesis is warranted.

It is not clear why there appears to be a stronger association between IGF2 ApaI genotype and FFM and function in women than in men in our study. The association between IGF2 ApaI genotype and SP was not significant for the men in cohort 1. Although A/A women demonstrated significantly lower leg strength during shortening contractions at 180°/s, and Kannus and Järvinen (25) have reported a significant correlation between leg strength during shortening contractions at this velocity and knee extensor power, isokinetic measures do not truly reflect power-generation capacity due to the factor of constant angular velocity. A lack of association between IGF2 polymorphism and muscle power may be expected given that IGF-II primarily affects muscle mass, but power generation involves the additional factor of voluntary neural drive (17). In contrast to our finding of no significant genotype association with grip strength, Sayer et al. (51) found significantly higher grip strength in G/G men compared with A/A men in a British cohort of middle-aged men and women. In addition, in a recent BLSA-based study of IGF2 ApaI genotype and body composition (49), our laboratory presented findings that were similarly inconsistent with a study by O'Dell et al. (41) that evaluated the same British cohort studied by Sayer et al. for the effect of IGF2 ApaI genotype on IGF-II protein levels and body composition. However, in both British studies, potential confounding variables, such as activity level, history of strength training, etc., were not accounted for as was done in this study.

Because the ApaI polymorphism is located within the promoter region of the human IGF2 gene, it likely affects transcription of IGF2, as demonstrated by the finding in mice that the IGF2 ApaI G allele was associated with higher mRNA levels than the A allele (55). Furthermore, variation within the IGF2 gene has been linked to elevated mRNA and protein levels of IGF-II and the associated development of double-muscling in some cattle breeds (15, 31). However, it is still unclear how specific IGF2 ApaI polymorphisms and other recently studied polymorphisms in the IGF2 gene region (14) may affect levels of IGF-II protein in serum and/or muscle to potentially influence ligand binding to the type I tyrosine kinase receptor and the development and/or loss of muscle mass and function with age in humans. O'Dell et al. (41) have reported that A/A men have higher serum IGF-II levels than G/G men. However, our laboratory did not show results consistent with this finding in a previous study (49). In fact, although the difference was not statistically different due to small sample size and high variability, 48 G/G men had 23.7% higher serum IGF-II levels compared with 10 A/A men. Given the above animal studies regarding the ApaI polymorphism (55), IGF2 expression and muscle mass (15, 31), and our limited finding regarding IGF-II serum levels above (49), one would expect that the IGF2 ApaI G/G subjects in the present study who have greater FFM and muscle strength may also have higher mRNA and serum IGF-II levels compared with A/A subjects. Collectively, the data available at the time of this writing clearly indicate the need to clarify these relationships across the adult age span before this information can be used to target individuals more susceptible to the effects of sarcopenia based on IGF2 and other candidate genes.

There are several limitations of the present study. First, we are showing the relationship between variation at only one gene locus and complex muscle phenotypes that are likely influenced by numerous genes and polymorphisms. Limited sample sizes, especially for cohort 1 data, prevented analysis of multi-locus genotypes in the present study. Also, small sample size and population stratification are the most likely explanations for deviation from Hardy-Weinberg equilibrium in men in cohort 2. In addition, although IGF-II plays a crucial role in muscle development (7), its role in the repair/regeneration process is less central and more secondary to other growth factors (i.e., IGF-I) (34). Thus multi-locus analysis with larger sample sizes should be performed in future studies.

In summary, the present study is the first to demonstrate an association between IGF2 genotype and strength and FFM in humans. IGF2 genotype appears to be associated with isokinetic arm and leg strength in men and women, but to a greater extent in women. Finally, our findings that the IGF2 genotype may be associated with FFM and strength at peak age (35 yr) and across the adult age span provide support for the hypothesis that genetic influences on developing muscle may affect muscle mass and function in later life.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. F. Hurley, Dept. of Kinesiology, Univ. of Maryland, College Park, MD 20742 (E-mail: benhur{at}umd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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